Proteins are typically synthesized with 20 amino acids, yet over 300 amino acids are found in proteins as a result of posttranslational modifications (PTMs). These natural non-genetically encoded amino acids (ngeAAs) modulate protein function and control fundamental cellular processes. Malfunction in PTM pathways leads to numerous human diseases including cancer, neurological and developmental disorders. Satisfactory genetic encoding of ngeAAs requires the development of efficient and accurate aminoacyl-tRNA formation and delivery to the ribosome by design of tRNAs, tRNA synthetases, and elongation factors that constitute orthogonal translation systems (OTSs). While some ngeAAs have been genetically encoded (e.g., N-acetyl lysine, phosphoserine (Sep)), OTSs have not been established for a number of critical PTMs. The overall goal of this proposal is to rewire translation by developing OTSs in Escherichia coli and mammalian cells for facile and precise production of natural and engineered proteins containing naturally occurring ngeAAs. This technology is essential to define the role of ngeAAs in cells and how their malfunction can cause disease. These general goals will be realized in three specific areas of the proposed work. I) We plan to create aminoacyl-tRNA synthetases for efficient synthesis of ngeAA-tRNA for a series of phosphoamino acids and lysine derivatives. Given the critical role of phosphorylation in cell signaling and the success of kinase inhibitors against cancer cells, and based on our success establishing an OTS for phosphoserine, we propose to establish OTSs for additional phosphoamino acids and their non-hydrolyzable analogs. We will further endeavor to develop OTSs for newly discovered lysine analogs that play important roles in gene expression and metabolism. II) Selenium, in the form of selenocysteine (Sec), is an essential trace element for human health, and selenoprotein mutations have been implicated in cancer and in conditions affecting the muscular, nervous, immune and endocrine systems. Site-directed insertion of Sec in E. coli and mammalian cells will allow production of natural, mutant and custom designed selenoproteins. We will also investigate the role of defective Sec biosynthesis in two severe human genetic diseases. III) Translational quality control by the elongation factors EF-Tu, EF1A, and EF-Sec will be studied along side efforts to design elongation factor variants that will specifically recognize and enhance co-translational incorporation of ngeAAs. The proposed work is significant because the ability to produce, purify, biochemically and structurally characterize proteins containing ngeAAs at defined sites is essential for elucidation of fundamental cellular processes. The innovation of the proposed work is to genetically encode these biologically relevant ngeAAs, and provide broad access to facile and efficient OTSs for biochemical and biomedical researchers, which will help unravel the complex network of PTMs and their role in human health and disease.